Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells.

Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation. While many drivers of HSC ageing have been proposed, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.

The mitochondrial metabolic checkpoint and aging of hematopoietic stem cells.

PURPOSE OF REVIEW: Cell-cycle checkpoints are surveillance mechanisms in eukaryotic cells that monitor the condition of the cell, repair cellular damages, and allow the cell to progress through the various phases of the cell cycle when conditions become favorable. We review recent advances in hematopoietic stem cell (HSC) biology, highlighting a mitochondrial metabolic checkpoint that is essential for HSCs to return to the quiescent state. RECENT FINDINGS: As quiescent HSCs enter the cell cycle, mitochondrial biogenesis is induced, which is associated with increased mitochondrial protein folding stress and mitochondrial oxidative stress. Mitochondrial unfolded protein response and mitochondrial oxidative stress response are activated to alleviate stresses and allow HSCs to exit the cell cycle and return to quiescence. Other mitochondrial maintenance mechanisms include mitophagy and asymmetric segregation of aged mitochondria. SUMMARY: Because loss of HSC quiescence results in the depletion of the HSC pool and compromised tissue regeneration, deciphering the molecular mechanisms that regulate the mitochondrial metabolic checkpoint in HSCs will increase our understanding of hematopoiesis and how it becomes dysregulated under pathological conditions and during aging. More broadly, this knowledge is instrumental for understanding the maintenance of cells that convert between quiescence and proliferation to support their physiological functions.

Mito-managing ROS & redox to reboot the immune system: Tapping mitochondria & redox management to extend the reach of hematopoietic stem cell transplantation.

Hematopoietic stem cells (HSCs) are responsible for life-long production of blood and immune cells. HSC transplantation (HSCT) is the original cell therapy which can cure hematological disorders but also has the potential to treat other diseases if technical and safety barriers are overcome. To maintain homeostatic hematopoiesis or to restore hematopoiesis during transplantation HSCs must perform both self-renewal, replication of themselves, and differentiation, generation of mature blood and immune cells. These are just two of the cell fate choices HSCs have; the transitional phases where HSCs undergo these cell fate decisions are regulated by reduction-oxidation (redox) signaling, mitochondrial activity, and cellular metabolism. Recent studies revealed that mitochondria, a key source of redox signaling components, are central to HSC cell fate decisions. Here we highlight how mitochondria serve as hubs in HSCs to manage redox signaling and metabolism and thus guide HSC fate choices. We focus on how mitochondrial activity is modulated by their clearance, biogenesis, dynamics, distribution, and quality control in HSCs. We also note how modulating mitochondria in HSCs can help overcome technical barriers limiting further use of HSCT.

Exploring Human Skin Aging at the Single-Cell Level.

In this issue of Developmental Cell, Zou et al. utilize eyelid samples to examine human skin aging at the single-cell level. They discover photo- and inflammation-related changes already in middle age and find that restoring youthful expression of KLF6 and HES1 may dial back some age-associated changes.

Inhibition of longevity regulator PAPP-A modulates tissue homeostasis via restraint of mesenchymal stromal cells.

Pregnancy-associated plasma protein-A (PAPP-A) is a secreted metalloprotease that increases insulin-like growth factor (IGF) availability by cleaving IGF-binding proteins. Reduced IGF signaling extends longevity in multiple species, and consistent with this, PAPP-A deletion extends lifespan and healthspan; however, the mechanism remains unclear. To clarify PAPP-A's role, we developed a PAPP-A neutralizing antibody and treated adult mice with it. Transcriptomic profiling across tissues showed that anti-PAPP-A reduced IGF signaling and extracellular matrix (ECM) gene expression system wide. The greatest reduction in IGF signaling occurred in the bone marrow, where we found reduced bone, marrow adiposity, and myelopoiesis. These diverse effects led us to search for unifying mechanisms. We identified mesenchymal stromal cells (MSCs) as the source of PAPP-A in bone marrow and primary responders to PAPP-A inhibition. Mice treated with anti-PAPP-A had reduced IGF signaling in MSCs and dramatically decreased MSC number. As MSCs are (1) a major source of ECM and the progenitors of ECM-producing fibroblasts, (2) the originating source of adult bone, (3) regulators of marrow adiposity, and (4) an essential component of the hematopoietic niche, our data suggest that PAPP-A modulates bone marrow homeostasis by potentiating the number and activity of MSCs. We found that MSC-like cells are the major source of PAPP-A in other tissues also, suggesting that reduced MSC-like cell activity drives the system-wide reduction in ECM gene expression due to PAPP-A inhibition. Dysregulated ECM production is associated with aging and drives age-related diseases, and thus, this may be a mechanism by which PAPP-A deficiency enhances longevity.

Mitochondrial Stress-Initiated Aberrant Activation of the NLRP3 Inflammasome Regulates the Functional Deterioration of Hematopoietic Stem Cell Aging.

Aging-associated defects in hematopoietic stem cells (HSCs) can manifest in their progeny, leading to aberrant activation of the NLRP3 inflammasome in macrophages and affecting distant tissues and organismal health span. Whether the NLRP3 inflammasome is aberrantly activated in HSCs during physiological aging is unknown. We show here that SIRT2, a cytosolic NAD+-dependent deacetylase, is required for HSC maintenance and regenerative capacity at an old age by repressing the activation of the NLRP3 inflammasome in HSCs cell autonomously. With age, reduced SIRT2 expression and increased mitochondrial stress lead to aberrant activation of the NLRP3 inflammasome in HSCs. SIRT2 overexpression, NLRP3 inactivation, or caspase 1 inactivation improves the maintenance and regenerative capacity of aged HSCs. These results suggest that mitochondrial stress-initiated aberrant activation of the NLRP3 inflammasome is a reversible driver of the functional decline of HSC aging and highlight the importance of inflammatory signaling in regulating HSC aging.

The mitochondrial unfolded protein response is activated upon hematopoietic stem cell exit from quiescence.

The mitochondrial unfolded protein response (UPRmt ), a cellular protective program that ensures proteostasis in the mitochondria, has recently emerged as a regulatory mechanism for adult stem cell maintenance that is conserved across tissues. Despite the emerging genetic evidence implicating the UPRmt in stem cell maintenance, the underlying molecular mechanism is unknown. While it has been speculated that the UPRmt is activated upon stem cell transition from quiescence to proliferation, the direct evidence is lacking. In this study, we devised three experimental approaches that enable us to monitor quiescent and proliferating hematopoietic stem cells (HSCs) and provided the direct evidence that the UPRmt is activated upon HSC transition from quiescence to proliferation, and more broadly, mitochondrial integrity is actively monitored at the restriction point to ensure metabolic fitness before stem cells are committed to proliferation.

Stem cell aging. A mitochondrial UPR-mediated metabolic checkpoint regulates hematopoietic stem cell aging.

Deterioration of adult stem cells accounts for much of aging-associated compromised tissue maintenance. How stem cells maintain metabolic homeostasis remains elusive. Here, we identified a regulatory branch of the mitochondrial unfolded protein response (UPR(mt)), which is mediated by the interplay of SIRT7 and NRF1 and is coupled to cellular energy metabolism and proliferation. SIRT7 inactivation caused reduced quiescence, increased mitochondrial protein folding stress (PFS(mt)), and compromised regenerative capacity of hematopoietic stem cells (HSCs). SIRT7 expression was reduced in aged HSCs, and SIRT7 up-regulation improved the regenerative capacity of aged HSCs. These findings define the deregulation of a UPR(mt)-mediated metabolic checkpoint as a reversible contributing factor for HSC aging.

Sirtuins, tissue maintenance, and tumorigenesis.

Aging is a degenerative process resulting in compromised tissue maintenance and increased susceptibility to diseases, such as cancer. Recent advancements support the notion that aging is a highly regulated process governed by evolutionarily conserved pathways. In mammals, tissue-specific adult stem cells (ASCs) persist throughout the lifetime to maintain and repair tissues. While reduced ASC self-renewal is thought to contribute to compromised tissue maintenance, increased self-renewal of cancer stem cells (CSCs) may lead to tumorigenesis. It is speculated that genetic regulators of aging, such as sirtuins, are likely to impinge upon the ASC compartments to regulate tissue maintenance and tumorigenesis. In this review, we discuss the emerging evidence linking sirtuins to normal and malignant ASC self-renewal, tissue maintenance, and tumorigenesis.

SIRT3 reverses aging-associated degeneration.

Despite recent controversy about their function in some organisms, sirtuins are thought to play evolutionarily conserved roles in lifespan extension. Whether sirtuins can reverse aging-associated degeneration is unknown. Tissue-specific stem cells persist throughout the entire lifespan to repair and maintain tissues, but their self-renewal and differentiation potential become dysregulated with aging. We show that SIRT3, a mammalian sirtuin that regulates the global acetylation landscape of mitochondrial proteins and reduces oxidative stress, is highly enriched in hematopoietic stem cells (HSCs) where it regulates a stress response. SIRT3 is dispensable for HSC maintenance and tissue homeostasis at a young age under homeostatic conditions but is essential under stress or at an old age. Importantly, SIRT3 is suppressed with aging, and SIRT3 upregulation in aged HSCs improves their regenerative capacity. Our study illuminates the plasticity of mitochondrial homeostasis controlling stem cell and tissue maintenance during the aging process and shows that aging-associated degeneration can be reversed by a sirtuin.

SIRT7 represses Myc activity to suppress ER stress and prevent fatty liver disease.

Nonalcoholic fatty liver disease is the most common chronic liver disorder in developed countries. Its pathogenesis is poorly understood, and therapeutic options are limited. Here, we show that SIRT7, an NAD(+)-dependent H3K18Ac deacetylase, functions at chromatin to suppress ER stress and prevent the development of fatty liver disease. SIRT7 is induced upon ER stress and is stabilized at the promoters of ribosomal proteins through its interaction with the transcription factor Myc to silence gene expression and to relieve ER stress. SIRT7-deficient mice develop chronic hepatosteatosis resembling human fatty liver disease. Myc inactivation or pharmacological suppression of ER stress alleviates fatty liver caused by SIRT7 deficiency. Importantly, SIRT7 suppresses ER stress and reverts the fatty liver disease in diet-induced obese mice. Our study identifies SIRT7 as a cofactor of Myc for transcriptional repression and delineates a druggable regulatory branch of the ER stress response that prevents and reverts fatty liver disease.

DNA-damage response in tissue-specific and cancer stem cells.

Recent studies have shown that tissue-specific stem cells (SCs) found throughout the body respond differentially to DNA damage. In this review, we will discuss how different SC populations sense and functionally respond to DNA damage, identify various common and distinct mechanisms utilized by tissue-specific SCs to address DNA damage, and describe how these mechanisms can impact SC genomic integrity by potentially promoting aging, tissue atrophy, and/or cancer development. Finally, we will discuss how similar mechanisms operate in cancer stem cells (CSCs) and can mediate resistance to chemo- and radiotherapy.

Hematopoietic stem cell quiescence promotes error-prone DNA repair and mutagenesis.

Most adult stem cells, including hematopoietic stem cells (HSCs), are maintained in a quiescent or resting state in vivo. Quiescence is widely considered to be an essential protective mechanism for stem cells that minimizes endogenous stress caused by cellular respiration and DNA replication. We demonstrate that HSC quiescence can also have detrimental effects. We found that HSCs have unique cell-intrinsic mechanisms ensuring their survival in response to ionizing irradiation (IR), which include enhanced prosurvival gene expression and strong activation of p53-mediated DNA damage response. We show that quiescent and proliferating HSCs are equally radioprotected but use different types of DNA repair mechanisms. We describe how nonhomologous end joining (NHEJ)-mediated DNA repair in quiescent HSCs is associated with acquisition of genomic rearrangements, which can persist in vivo and contribute to hematopoietic abnormalities. Our results demonstrate that quiescence is a double-edged sword that renders HSCs intrinsically vulnerable to mutagenesis following DNA damage.